Seeds are normally the product of the sexual process. The typical seed consists of an embryo in a resting phase, incorporating and surrounded by nutritive tissues which provides for food storage and contained within a protective coat.
Some plants will regenerate asexually also. Asexual embryogenesis in vivo is a well known phenomenon in the Rustaceae, Coctaceae, Celastraceae, Liliaceae, Myrtaceae, Orchidaceae, Rosaceae and Solanaceae families. Tisserat, et al, Horticultural Reviews, pp. 1-99 (1979).
It is possible to produce asexual embryos in vitro. Techniques for the regeneration of plants from tissue cultures have been developed for a variety of species. This technology can be used as a means of cloning plants on a commercial scale. This would have application in processes where it is undesirable to allow the selected plants to undergo meiosis and genetic recombination to produce seeds such as in hybrid production, in propagation of elite germplasm in species which produce seeds infrequently, or in the "fixation" of genetic traits in obligate cross pollinating species. A cell from almost any part of a plant can produce an asexual embryo and from it regenerate the entire plant.
Such artificial "seeds" would substantially benefit the seed industry, agriculture, forestry and horticulture. Specific applications of such techniques include the following:
1) as a breeding tool to reduce the number of generations required to reach a seed of commerce;
2) as a means of producing hybrid seed through the propagation of parental plants such as male and female sterile plants;
3) as a means of propagating valuable agronomically superior plants for the direct establishment of production fields;
4) as a means of storing a plant in a quiescient state to synchronize planting, for transport in a sterile condition, or for long-term germplasm storage;
5) as a means of producing seed of species which flower and produce viable seed only sporadically (e.g. bamdoo).
In vitro asexual embryogenesis techniques produce an embryo, which is identical in most respects to a normal zygotic embryos. These embryos lack a surrounding endosperm and the latter being derived exclusively from maternal tissue in the zygotic seed. Another distinct difference between the two types of embryos is that asexual embryos cannot be dried and stored for any period of time. There are two ways to protect the embryos from drying: they can be coated with compounds which prevent the loss of water (i.e. avoid drying) or alternatively, they can be induced to tolerate drying by synthesizing protective chemicals which protect the cells from the consequences of water loss. Previous methods have emphasized ways of reducing water loss and certain coating techniques have been developed. Edward W. Janssen describes in his U.S. Pat. No. 3,920,436 a process that creates an artificial protective environment for plants by coating a portion of the plant with a fluid agent comprising hydrophilic urethane prepolymer. However, this system is designed for the protection of mature plants by preventing water loss and since the prepolymer is insoluble, it would not allow the embryo to germinate.
Wet capsules have been used to replace the seed coat. Plant Genetics Inc. (PGI) has developed a process for gel coating somatic embryos to help withstand the rigors of handling and field planting. The coating is comprised of two parts: a matrix containing essential nutrients etc. and a protective polymer seed coat which prevents desiccation. The resulting capsules are about the size of soya bean seeds, spherical in shape and thus adaptable to large scale agricultural use. However, there are disadvantages in such a wet capsule. The capsules need special low temperature storage conditions otherwise they will start to germinate prematurely making long-term storage impossible. The vigor of seedlings may also be reduced due to the avoidance of the natural drying process.
Janick and Kitto describe in their U.S. Pat. No. 4,615,141 certain methods for enhancing desiccation tolerance in somatic embryos The asexual embryos are "hardened" by various means during their development to induce resistance to environmental stress. The hardened asexual embryos are then coated with a solution of non-toxic biocompatible, water soluble synthetic coating material. The resulting solution-coated embryos are then dried to provide viable embryos encapsulated in the coating material. Embryos encapsulated by this method are relatively short-lived in the dry state (ca. 2-4 days).
Janick and Kitto described four separate hardening treatments: high inoculum density, high sucrose concentrations, chilling and/or exposure to abscisic acid (ABA). Each of these pretreatments enhances the viability of the embryos after encapsulation. These treatments increase synthesis of ABA, a plant growth regulator. It would appear that ABA is a factor in the survival of somatic embryos. The type of hardening treatment used is critical since it renders asexual embryos quiescent and induce the endogenous synthesis of specific protectants, such as oxygen-free radical scavengers, thereby increasing the survival rate of the embryos following desiccation.
Tolerance of desiccation has at least two components. The first is tolerance to the loss of water, and the second is the ability to maintain viability during prolonged periods of dryness. Tolerance to the loss of water can be induced in plant tissues by the induction of quiescence or dormancy. Tolerance of prolonged storage is provided in part by high levels of lipid soluble free radical scavenging systems (antioxidants), such as tocopherol. Both components of desiccation can be induced by ABA, but the response of the embryo is dependent on the stage of development at the time of treatment. Quiescence can be induced at a wide range of developmental stages, but the induction of antioxidant synthesis is accomplished only at specific stages. In the method of Janick and Kitto, the ABA treatment was not applied at the correct stage of development to induce synthesis of antioxidants. This would explain why their embryos could not survive drying without coating and why the coated embryos survived only 2 to 4 days.